Project Summary Necrotizing enterocolitis in very low birth weight infants remains a major challenge. Our reactive clinical approach to necrotizing enterocolitis has changed little in four decades. Morbidity and mortality from necrotizing enterocolitis remain unacceptably high, and preventive strategies rely on interventions with limited, if any, efficacy. We have assembled an extensive set of specimens, accompanied by rich clinical metadata, with which to define mechanisms that underlie the development of necrotizing enterocolitis in very preterm infants. Our proposal extends our recently published findings that a gut bacterial dysbiosis, characterized by over-representation of Gammaproteobacteria and under-representation of obligate anaerobic bacteria, precede, and plausibly contribute to, the development of necrotizing enterocolitis. Our overarching hypothesis is that identifiable microbial and/or host mechanisms signify an infant at risk for necrotizing enterocolitis. Our long-term goals are to use our knowledge of microbial-driven pathophysiology in necrotizing enterocolitis to build interventions to prevent the assembly of dysbiotic gut bacterial populations, to characterize the host biology prior to the sudden appearance of necrotizing enterocolitis. By generating these data, we hope to lessen the severity of necrotizing enterocolitis, or to prevent it altogether. Our Specific Aims are: (1) Conduct a comprehensive genomic analysis of gut bacteria prior to necrotizing enterocolitis onset, and (2) Define the gut biology and host response prior to necrotizing enterocolitis onset. All specimens from cases will be those obtained before this event occurred. Control materials will be matched to cases, according to gestational age at birth, and day of life specimens were produced. To accomplish these Aims, we will use our archive of pre-event stools and sera/plasmas from 977 very low birthweight infants from three neonatal intensive care units. Forty-six of these infants experienced NEC, after excluding those who also had congenital heart disease. We will employ genomic characterization of isolates, transcriptomics, metabolomics, viromics, and immunoproteomics in coordinated analyses to illuminate microbial and host biology underlying necrotizing enterocolitis. We will also interrogate populations of bacteria associated with protection from necrotizing enterocolitis. All studies will initially analyze a primary cohort from St. Louis, and then validate findings in specimens from Oklahoma City and Louisville. These case-control and time-series comparisons will provide mechanistic insight into an intractable and highly consequential complication of preterm birth, using an exceptionally relevant system, i.e., the colonized human infant. By project conclusion, we will generate mechanistically-informed data to guide attempts to better anticipate, treat, or, ideally, prevent, necrotizing enterocolitis, a devastating complication of preterm birth.